PROJECT SUMMARY/ABSTRACT Thirty years ago Hart and colleagues discovered that many nuclear and cytoplasmic proteins are modified with b-linked N-acetylglucosamine (O-GlcNAc), overturning a paradigm about where glycosylated proteins are found in eukaryotes. Since then, O-GlcNAc modifications, which are installed by the enzyme O-GlcNAc transferase (OGT), have been identified on both metabolic and cell signaling enzymes, transcription factors, epigenetic regulators, and scaffolding proteins. O-GlcNAc levels increase when glucose levels are high, and perturbations in protein O-GlcNAcylation has been linked to diseases caused by protein misregulation, such as cancer and Alzheimer?s. It has been speculated that methods to reduce O-GlcNAcylation on targeted substrates would be therapeutically advantageous. Developing such methods first requires understanding of how OGT chooses its substrates. To date no substrate consensus sequence has been identified because substrate selection does not occur at the active site. However, OGT?s N-terminus contains a unique 13.5 tetratricopeptide repeat (TPR) region that has been implicated in substrate selection. Despite its presumed role in substrate regulation, there is minimal insight into a mechanism by which OGT substrates bind to the TPRs or whether different subsets of substrates bind via unique interactions or distinct mechanisms. I propose experiments to elucidate how OGT chooses its substrates by investigating two conserved ladder motifs within the TPR lumen that we hypothesize are critical for substrate recognition and selectivity. In Aim 1, I will make TPR-domain mutants to the conserved aspartate ladder that we hypothesize will alter binding to substrate side chains and use both in vitro glycosylation assays and glycosite mapping proteomics to globally determine the role of this ladder in substrate selectivity. In Aim 2, I will make mutants to the conserved asparagine ladder that we hypothesize will alter binding to the backbone of substrates and use in vitro glycosylation assays along with covalent chemical capture techniques to determine how the asparagine ladder is involved in substrate recognition and the directionality of substrate engagement by the TPRs of OGT. In Aim 3, I will develop an approach to investigate how changes in OGT substrate selectivity affects cellular phenotypes (e.g., viability and mitochondria structure) of mammalian cells. This approach will make it possible to link the in vitro studies, which provide information on substrate alterations of mutations, to cellular function. Results from this study will be the first to define the role of the TPRs in OGT substrate selection and provide an extended consensus sequence for substrate selection. This information will enable the advancement of new strategies to selectively interrogate O- GlcNAc?s role on specific substrates and the development of selective inhibitors targeting specific substrates without pursuing the active site.